Analysis of GET pathway receptors in Arabidopsis thaliana

Abstract

Tail-anchored (TA) proteins are a class of integral membrane proteins that are involved in diverse cellular processes, including protein translocation (SEC61), vesicle trafficking (SNAREs) and apoptosis (BCL-2). A transmembrane domain (TMD) at the extreme C-terminus prevents TA proteins access to the classical co-translational route into the endoplasmic reticulum (ER); instead, they require post-translational insertion via the Guided Entry of Tail-anchored proteins (GET) pathway that was recently described in yeast and mammals. Here, the cytosolic targeting factor GET3 (in yeast; TRC40 in mammals) chaperones newly synthesized TA proteins from the ribosome to the ER where the GET1-GET2 (in yeast; WRB-CAML in mammals) receptor complex facilitates membrane insertion. However, so far hardly anything is known about the conservation and function of the GET pathway in higher plants.

We identified several GET components in Arabidopsis thaliana through in silico analysis, including an orthologue of GET1, and found a role of the GET pathway in root hair elongation. Additionally, direct in planta interaction analysis using immunoprecipitation-mass spectrometry of AtGET1-GFP expressing lines identified a promising candidate for the membrane receptor GET2, which has no sequence orthologue in plants. Remarkably, this protein resembles a similar TMD architecture as GET2 and CAML and carries a conserved positively charged motif in its N-terminus. We provide here experimental evidence that this protein of unknown function might indeed represent the functional GET2 orthologue in Arabidopsis thaliana.